Antibody affinity and specificity are widely applied in a broad range of fields, including therapeutic applications, diagnoses, and reagents. With recent years' advances in antibody engineering, miniaturized antibodies in a wide variety of forms, such as scFv (single chain Fv) and diabody have been reported [Bird et al., Science (1988), vol. 242, pp. 423-426; Holliger et al., Proc. Natl. Sci. USA (1993), vol. 90, pp. 6444-6448]. Furthermore, artificial antibody molecules that are smaller and more stable than antibodies have been developed [Skerra, Curr. Opin. Biotechnol. (2007), vol. 18, pp. 295-304].
For example, JP-T-2001-500531 discloses an artificial antibody of a fibronectin III type domain that mimics the CDR (complementarity determining region) of an antibody. This artificial antibody has an antigen recognition site artificially created on the surface of a protein by randomizing flexible loops on the protein surface.
RNF8 is an E3 ubiquitin ligase having a RING-Finger domain and an FHA domain, and is known to be involved in responses to DNA damage. Conformational analysis showed that the FHA domain of RNF8 (PDB code: 2PIE, 2CSW) has an Immunoglobulin(Ig)-like structure (β sandwich structure) with five loops (two long loops and three short loops) on the protein surface (FIG. 1). Also, the binding to the binding partner MCD1 has been shown to occur via the two long loops in the FHA domain. This binding pattern is similar to the antigen recognition pattern of antibody; the FHA domain of RNF8 is expected to have a suitable structure for development of artificial antibody molecules.
Also, RNF8 is an intracellular protein and an artificial antibody derived from RNF8 is therefore expected to work as a highly functional intrabody (intracellularly expressed antibody). An intrabody is defined as an antibody (primarily scFv) that works within cells to bind to an intracellular protein as a target thereof. Intrabodies have been developed for therapeutic uses for a wide variety of disease, for example, AIDS, cancers, Alzheimer's disease, Parkinson's disease, and Huntington's disease.
Furthermore, intrabodies are effective in knocking out intracellular targets. RNAi is a generally known knock-out form of intracellular target. However, RNAi is not applicable to analyzing post-translationally modified targets because the gene is knocked out as it is, and also because the half-life of the intracellular RNA is short. Intrabodies have longer half-lives in cells than those of RNA, and can knock out at the protein level. For this reason, intrabodies make it possible to solve the above-described problem with RNAi [Zhou et al., Mol. Cell. (2000), vol. 6, pp. 751-756; Jendreyko et al., J. Biol. Chem. (2003), vol. 278, pp. 47812-47819; Melchionna et al., J. Mol. Biol. (2007), vol. 374, pp. 641-654].
However, the binding activity often decreases or disappears completely when the antibody is expressed as an intrabody in cells under reducing environmental conditions due to the disruption of disulfide bonds in their molecular structure.
Initial attempts to solve this problem included the development of an antibody deprived of intramolecular cysteine involved in disulfide bonds [Proba et al., J. Mol. Biol. (1997), vol. 265, pp. 161-172] and an antibody with increased stability [Ohage et al., J. Mol. Biol. (1999), vol. 291, pp. 1129-1134]. As a method of preparing a functional antibody in cells more effectively, a method of direct selection of scFv in cells was developed [Visintin et al., Proc. Natl. Acad. Sci. USA (1999), vol. 96, pp. 11723-11728]. However, selection systems using cells have limitations with regard to ligation efficiency of antibody library onto the vector and cell transformation efficiency. For this reason, it is difficult to apply selection systems using cells to a library with greater diversity. Therefore, combinations with another method such as phage display or ribosome display, and a method of validation for selected scFv have been proposed.